Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca2+ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca2+ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca2+ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca2+ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca2+ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.

The addition and removal of presynaptic terminals reconfigures neuronal circuits of the mammalian neocortex, but little is known about how this presynaptic structural plasticity is controlled. Since mitochondria can regulate presynaptic function, we investigated whether the presence of axonal mitochondria relates to the structural plasticity of presynaptic boutons in mouse neocortex. We found that the overall density of axonal mitochondria did not appear to influence the loss and gain of boutons. However, positioning of mitochondria at individual presynaptic sites did relate to increased stability of those boutons. In line with this, synaptic localization of mitochondria increased as boutons aged and showed differing patterns of localization at en passant and terminaux boutons. These results suggest that mitochondria accumulate locally at boutons over time to increase bouton stability.

The brains of higher organisms are composed of anatomically and functionally distinct regions performing specialised tasks; but regions do not operate in isolation. Orchestration of complex behaviours requires communication between brain regions, but how neural activity dynamics are organised to facilitate reliable transmission is not well understood. We studied this process directly by generating neural activity that propagates between brain regions and drives behaviour, allowing us to assess how populations of neurons in sensory cortex cooperate to transmit information. We achieved this by imaging two hierarchically organised and densely interconnected regions, the primary and secondary somatosensory cortex (S1 and S2) in mice while performing two-photon photostimulation of S1 neurons and assigning behavioural salience to the photostimulation. We found that the probability of perception is determined not only by the strength of the photostimulation signal, but also by the variability of S1 neural activity. Therefore, maximising the signal-to-noise ratio of the stimulus representation in cortex is critical to its continued propagation downstream. Further, we show that propagated, behaviourally salient activity elicits balanced, persistent, and generalised activation of the downstream region. Hence, our work adds to existing understanding of cortical function by identifying how population activity is formatted to ensure robust transmission of information, allowing specialised brain regions to communicate and coordinate behaviour.

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10Addition and removal of presynaptic terminals reconfigures neuronal circuits of the 11 mammalian neocortex, but little is known about how this presynaptic structural plasticity is 12 controlled. Since mitochondria can regulate presynaptic function, we investigated whether the 13 Figure 1 -Tracking bouton plasticity and mitochondrial positioning in axons of motor cortex 1 neurons. (A) AAV expressing cytosolic EGFP and a mitochondrial targeting sequence (MTS) 2 conjugated to TagRFP was injected in to M1/M2 and a glass cranial window was implanted over 3 S1. (B) A series of coronal brain slices showing viral injection site across the M1/M2 border (inset, 4 top) and the axonal projection site at S1 under the cranial window (inset, bottom). Only the 5 ipsilateral half of brain sections are shown. Pr = parietal cortex, D = dorsal, L = lateral, A = anterior. 6 (C) Cropped 2P images from in vivo imaging show axons with high EPB (EPB-rich) or TB (TB-rich) 7 densities. (D) (top) Imaging timeline for tracking bouton structural plasticity (loss and gain). Viral 8 injection and cranial window implantation were performed 24 days prior to initial 2P imaging. 9 Arrowheads indicate imaging timepoints. (bottom) Structure and mitochondrial localisation in a 10 single cropped axon over 35 days imaged using in vivo 2P microscopy. Some boutons are labelled 11 with arrowheads to show examples of stable (yellow), lost (red) or gained (green) boutons. (E) 12Gaussian mixture modelling (GMM) was used to determine two potential populations (EPB-rich 13and TB-rich) that result in the observed sample distribution of EPB and TB densities for each 14 axonal segment (mean across time). Axons that had posterior probabilities below 70% were not 15 assigned to a group (circled; see Methods). Contour lines indicate the slope of the GMM 16 distribution. 17 18 A substantial projection from the motor cortex is made up of long-range axons that ramify in 19